Lineage in patterning the developing limb and heart

描述(申请人提供)：这项建议的主要目的是阐明发育中的肢体和心脏中不同细胞类型之间的谱系关系。将使用一种强大的技术来确定雏鸡肢体中的血统关系，该技术涉及将胚胎前体细胞与逆转录病毒载体库一起注射。每个载体都带有一个不同的“标签”，随后对注射细胞的标签进行聚合酶链式反应测序，用来确定后期肢芽或成熟肢体中克隆相关的单个细胞。肢体的这一分析需要解决的关键问题包括：肢芽内各种细胞类型的具体化和谱系关系的时间，肢芽中是否有明显的背侧和腹侧隔室，以及肢芽内指定近端肢体节段的时间。我们还将对心脏瓣膜不同部分的谱系进行分析。目前认为心脏瓣膜起源于心内膜分层，形成一种称为心垫的前体群体。然而，我们有迹象表明，这些垫子可能由3个不同的细胞群组成，每个细胞群在成熟瓣膜中有不同的命运。这将在小鼠胚胎中使用基于重组酶的命运映射进行测试。除了解决肢体和心脏发育的具体问题外，这些研究还将为设计未来的实验提供重要的背景。最后，这些研究中产生的试剂也将使我们能够明确地测试肢体芽中的肌腱和软骨前体是否是专用的前体细胞，或者它们是否有助于肢体中的其他细胞类型。此外，同样的试剂将用于测试肌腱和骨骼祖细胞是否需要自主表达HOX基因，以正确形成肢体模式。

DESCRIPTION (provided by applicant): The major objective of this proposal is to elucidate the lineage relationships between the different cell types in the developing limb and heart. Lineage relationships in the limb will be determined in the chick using a powerful technique involving injection of embryonic progenitors with a library of retroviral vectors. Each vector carries a distinct "tag" and subsequent PCR sequencing of the tag from injected cells is used to define individual cells in the later limb bud or mature limb that are clonally related. Critical questions to be addressed by this analysis in the limb include the timing of specification and lineage relationships of various cell types within the limb bud, whether there are distinct dorsal and ventral compartments in the limb bud, and the time when proximodistal limb segments are specified within the limb bud. We will also conduct an analysis of the lineages contributing to various portions of the valves of the heart. Heart valves are currently thought to derive from delanination of the endocardium, forming a precursor population called the cardiac cushion. However, we have indications that the cushions may form from 3 distinct cell populations, each with different fate in the mature valve. This will be tested using recombinase-based fate mapping in mouse embryos. In addition to addressing specific questions in limb and heart development, these studies will provide an important context for designing future experiments. Finally, the reagents generated in these studies will also allow us to definitely test whether the tendon and cartilage precursors in the limb bud are dedicated progenitors or whether they contribute to other cell types in the limb. Moreover, the same reagents will be used to test whether Hox gene expression is required autonomously in the tendon and skeletal progenitors for proper patterning of the limb.